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The crystal structure of orthorhombic Bovine Pancreatic Ribonuclease A has been determined to 0.85Å resolution using low temperature, 100K, synchrotron X-ray data collected at 16000keV (λ = 0.77Å). This is the first ultra-high-resolution structure of a native form of Ribonuclease A to be reported. Refinement carried out with anisotropic displacement parameters, stereochemical restraints, inclusion of H atoms in calculated positions, five SO42− moieties, eleven ethanol molecules and 293 water molecules, converged with final R values of R1(Free) = 0.129 (4279 reflections) and R1 = 0.112 (85346 reflections). The refined structure was deposited in the Protein Data Bank as structure 7p4r. Conserved waters, using four high resolution structures, have been investigated. Cluster analysis identified clusters of water molecules that are associated with the active site of Bovine Ribonuclease A. Particular attention has been paid to making detailed comparisons between the present structure and other high quality Bovine Pancreatic Ribonuclease A X-ray crystal structures with special reference to the deposited classic monoclinic structure 3RN3 Howlin et al [1]. Detailed studies of various aspects of hydrogen bonding and conformation have been carried out with particular reference to active site residues Lys-1, Lys-7, Gln-11, His-12, Lys-41, Asn-44, Thr-45, Lys-66, His-119 and Ser-123. For the two histidine residues in the active site the initial electron density map gives a clear confirmation that the position of His-12 is very similar in the orthorhombic structure to that in 3RN3. In 3RN3 His-119 exhibited poor electron density which was modelled and refined as two distinct sites, A (65%) and B (35%) but with respect to His-119 in the present ultra-high resolution orthorhombic structure there is clear electron density which was modelled and refined as a single conformation distinct from either conformation A or B in 3RN3. Other points of interest include Serine-32 which is disordered at the end of the sidechain in the present orthorhombic form but has been modelled as a single form in 3RN3. Lysine-66: there is density indicating a possible conformation for this residue. However, the density is relatively weak, and the conformation is unclear. Three types of amino acid representation in the ultra-high resolution electron density are examined: (i) sharp with very clearly resolved features, for example Lys-37; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry, for example Tyr-76; (iii) poor density and difficult or impossible to model, an example is Lys-31 for which density is missing except for Cβ. The side chains of Gln-11, His-12, Lys-41, Thr-45 and His-119 are generally recognised as being closely involved in the enzyme activity. It has also been suggested that Lys-7, Asp-44, Lys-66, Phe-120, Asp-121 and Ser-123 may also have possible roles in this mechanism. A molecular dynamics study on both structures has investigated the conformations of His-119 which was modelled as two conformations in 3RN3 but is observed to have a single clearly defined conformation in the present orthorhombic structure. MD has also been used to investigate Lys-31, Lys-41 and Ser32. The form of the Ribonuclease A enzyme used in both the present study and in 3RN3 [1] includes a sulphate anion which occupies approximately the same location as the PO42− phosphate group in protein nucleotide complexes [2]. The present structure contains 5 SO42− groups SO41151 – SO41155 two of which, SO41152 and SO41153 are disordered, SO41152 being in the active site, and 11 EtOH molecules, EOH A 201 – EOH A 211 all of which have good geometry. H atoms were built into the EtOH molecules geometrically. Illustrations of these features in the present structure are included here. The sulphates are presumably present in the material purchased for use in the present study. 293 water molecules are included in the present structure compared to 134 in 3RN3[1].

Multiple active vaccination approaches have proven ineffective in reducing the substantial morbidity and mortality caused by respiratory syncytial virus (RSV) in infants and older adults (aged ≥65 years). A vaccine conferring a substantial and sustainable boost in neutralising activity is required to protect against severe RSV disease. To that end, we evaluated the safety and immunogenicity of DS-Cav1, a prefusion F subunit vaccine.In this randomised, open-label, phase 1 clinical trial, the stabilised prefusion F vaccine DS-Cav1 was evaluated for dose, safety, tolerability, and immunogenicity in healthy adults aged 18-50 years at a single US site. Participants were assigned to receive escalating doses of either 50 μg, 150 μg, or 500 μg DS-Cav1 at weeks 0 and 12, and were randomly allocated in a 1:1 ratio within each dose group to receive the vaccine with or without aluminium hydroxide (AlOH) adjuvant. After 71 participants had been randomised, the protocol was amended to allow some participants to receive a single vaccination at week 0. The primary objectives evaluated the safety and tolerability at every dose within 28 days following each injection. Neutralising activity and RSV F-binding antibodies were evaluated from week 0 to week 44 as secondary and exploratory objectives. Safety was assessed in all participants who received at least one vaccine dose; secondary and exploratory immunogenicity analysis included all participants with available data at a given visit. The trial is registered with ClinicalTrials.gov, NCT03049488, and is complete and no longer recruiting.Between Feb 21, 2017, and Nov 29, 2018, 244 participants were screened for eligibility and 95 were enrolled to receive DS-Cav1 at the 50 μg (n=30, of which n=15 with AlOH), 150 μg (n=35, of which n=15 with AlOH), or 500 μg (n=30, of which n=15 with AlOH) doses. DS-Cav1 was safe and well tolerated and no serious vaccine-associated adverse events deemed related to the vaccine were identified. DS-Cav1 vaccination elicited robust neutralising activity and binding antibodies by 4 weeks after a single vaccination (p0·0001 for F-binding and neutralising antibodies). In analyses of exploratory endpoints at week 44, pre-F-binding IgG and neutralising activity were significantly increased compared with baseline in all groups. At week 44, RSV A neutralising activity was 3·1 fold above baseline in the 50 μg group, 3·8 fold in the 150 μg group, and 4·5 fold in the 500 μg group (p0·0001). RSV B neutralising activity was 2·8 fold above baseline in the 50 μg group, 3·4 fold in the 150 μg group, and 3·7 fold in the 500 μg group (p0·0001). Pre-F-binding IgG remained significantly 3·2 fold above baseline in the 50 μg group, 3·4 fold in the 150 μg group, and 4·0 fold in the 500 μg group (p0·0001). Pre-F-binding serum IgA remained 4·1 fold above baseline in the 50 μg group, 4·3 fold in the 150 μg group, and 4·8 fold in the 500 μg group (p0·0001). Although a higher vaccine dose or second immunisation elicited a transient advantage compared with lower doses or a single immunisation, neither significantly impacted long-term neutralisation. There was no long-term effect of dose, number of vaccinations, or adjuvant on neutralising activity.In this phase 1 study, DS-Cav1 vaccination was safe and well tolerated. DS-Cav1 vaccination elicited a robust boost in RSV F-specific antibodies and neutralising activity that was sustained above baseline for at least 44 weeks. A single low-dose of pre-F immunisation of antigen-experienced individuals might confer protection that extends throughout an entire RSV season.The National Institutes of Allergy and Infectious Diseases.

Stroke can cause significant disability and impact quality of life. Multidisciplinary neurorehabilitation that meets individual needs can help to optimise recovery. Rehabilitation is essential for best quality care but should start early, be ongoing and involve effective teamwork. We describe current stroke rehabilitation processes, from the hyperacute setting through to inpatient and community rehabilitation, to long-term care and report on which UK quality care standards are (or are not) being met. We also examine the gap between what stroke rehabilitation is recommended and what is being delivered, and suggest areas for further improvement.

This article presents a reinterpretation of John Locke's contribution to debates about the interest rate in the seventeenth century. It suggests that his argument that England should maintain the ‘natural’ rate, rather than impose a lower rate, was motivated by his theological, moral, and social conceptions of credit and its dependence on trust. In order to solve the endemic shortage of metal coin limiting the growth of monetary exchange in England, Locke stressed that the higher, ‘natural’ rate of interest would facilitate interpersonal borrowing and lending among neighbours, allowing currency to flow more freely around the country. By contrast, while he acknowledged that institutional creditors such as goldsmith-bankers could quicken the circulation of money by issuing debt instruments like bills of exchange, he saw institutional credit as a threat to the moral community. Not only did he question how people could rationally trust financiers without any epistemic apprehension of their personal probity, but he moreover doubted whether individuals accumulating so much money were likely to act trustworthily. Finally, using an otherwise unstudied dialogue about the Bank of England, this article argues Locke extended his criticisms about the threats posted by private banks to the country's nascent system of public credit.

This article recovers mid-century proposals for sciences of concord and contextualizes them as part of a broader politics of commercial knowledge in eighteenth-century Britain. It begins by showing...

Ray resections have been a viable treatment option for patients with tumors, trauma, infection, vascular insufficiency, or other abnormalities of the hand since the procedure was described in the 1920s. The creation of a functional hand after central ray resection presents unique technical challenges: insufficient closure of the gap between the metacarpals bordering the resected ray can produce an enlarged space between remaining digits and potentially cause digital malrotation, both of which negatively affect hand function. The goal is to make the space between resulting fingers as close to normal as possible. A number of procedures were described to address this issue, but unfortunately, they can be technically onerous and may require prolonged immobilization, the use of internal hardware, or the use of temporary hardware requiring removal.We describe a technique for amputation of the affected ray at the proximal metacarpal metadiaphyseal flare and a concomitant closing wedge osteotomy to allow superior gap closure between the residual fingers while maintaining the structure of the carpus and alignment of the hand. This improves functional and aesthetic outcomes after central ray resection of the hand. Key words: Metacarpal ray resection, Middle ray amputation, Ray amputation hand

The global disruption caused by the COVID-19 pandemic has impacted every sector in society. Education has been affected at all levels, from kindergarten to Continuing Professional Development, as i...

Recent literature has demonstrated the importance of fieldwork in geology. However, as resources become scarce, field experiences are often targeted for cuts. This was the case at the University of...

Cone penetration testing (CPT) is a preferred method for characterizing soil profiles for evaluating seismic liquefaction triggering potential. However, CPT has limitations in characterizing highly stratified profiles because the measured tip resistance ( q c ) of the cone penetrometer is influenced by the properties of the soils above and below the tip. This results in measured q c values that appear “blurred” at sediment layer boundaries, inhibiting our ability to characterize thinly layered strata that are potentially liquefiable. Removing this “blur” has been previously posed as a continuous optimization problem, but in some cases this methodology has been less efficacious than desired. Thus, we propose a new approach to determine the corrected q c values (i.e. values that would be measured in a stratum absent of thin-layer effects) from measured values. This new numerical optimization algorithm searches for soil profiles with a finite number of layers which can automatically be added or removed as needed. This algorithm is provided as open-source MATLAB software. It yields corrected q c values when applied to computer-simulated and calibration chamber CPT data. We compare two versions of the new algorithm that numerically optimize different functions, one of which uses a logarithm to refine fine-scale details, but which requires longer calculation times to yield improved corrected q c profiles.

Jon Cooper and Ashley Borthwick of law firm Womble Bond Dickinson summarise the key workplace safety guidance for the UK construction industry as Covid-19 restrictions are eased.

Multiple interbedded fine-grained layers in a sand deposit have a “smoothing” effect on the measured Cone Penetration Test (CPT) tip resistance (qc), resulting in a significant underestimation of the predicted liquefaction resistance of the sand layers. Trends identified by De Lange [14] through calibration chamber tests on stratified sand-clay profiles are used herein to develop a new thin-layer correction procedure for qc (the “Deltares” procedure). The efficacies of the Deltares and the independently-developed Boulanger and DeJong [6] procedures are both directly assessed using CPT data from calibration chamber tests and indirectly inferred from CPT-based liquefaction case histories in Christchurch, New Zealand. The results highlight limitations of the assessed thin-layer CPT qc correction procedures for layers less than 40 mm thick. Multiple, interbedded thin layers also influence the measured CPT sleeve friction (fs), but in a more complex way than they influence qc. To-date, no procedures have been proposed to address all the thin-layer-effects phenomena on the measured fs, with errors in properly characterizing the fs of a layer inherently influencing the accuracy of predicting the liquefaction susceptibility and potential of the layer. In totality, the thin-layer-effects correction procedures proposed to-date generally result in slightly less accurate predictions of the observed liquefaction severity for cases having highly stratified profiles, opposite of what would be expected and desired.

The innovations of the twenty-first century in digital technology and media have had major influences in the way young urbanites and future city designers think as well as experience places. In embracing this conceptual shift of how new communication technologies are changing people’s perceptions, experiences and ways of knowing the city, Oxford Brookes University continues to develop pedagogical methods which use new media in the teaching, learning and production of contemporary urban design.These methods are examined in this paper in the context of three undergraduate modules:• Taking the urban design learning experience out of the classroom and into the field using mobile lectures;• Using digital media in research and engaging with oral, visual and sensory experiences;• Developing design communication skills using video, animation, interactive PDFs and website design;• Bringing new perspectives to classic theoretical concepts by engaging with different media: Cullen’s Townscape from serial expe...

The use of waters of impaired quality has been suggested as a means to expand available water resources supply for water-limited communities. An ongoing concern is the safety of supplies that use wastewater because of the potential for introduction of emerging contaminants such as pharmaceuticals into drinking water supplies. Prior research into contaminants of emerging concerns (CECs) have included a variety of methods, but the only consistent removal is with reverse osmosis (RO) membranes, ultraviolet light (UV), and advanced oxidation processes (AOP). However, few of these prior studies have measurable quantities of these contaminants in the influent wastewater, so determining actual removal percentages is difficult. This project was designed to evaluate the removal of CECs to verify that a 3-log removal of common constituents was realized. Spike testing was used to compare to prior research and to evaluate whether the project costs were competitive with other forms of reuse or other water supplies. The combination of RO/UV/AOP was effective at obtaining a 3-log removal of CECs, but the RO and UV/AOP processes alone were not capable of removing all substances. However, despite the extensive treatment, the proposed process was both competitive cost-wise and met the water quality goals.

The City of Pembroke Pines is embarking on an alternative water supply (AWS) project that includes the potential of using treated wastewater for aquifer recharge. The concept includes the use of reverse osmosis membranes, ultraviolet disinfection and advanced oxidation processes downstream of activated sludge and microfiltration. One of the problems is that the permeate leaves the process grossly under-saturated, because with respect to minerals, virtually everything in the water is removed by the reverse osmosis membranes. The practical natural minimum hardness level for water is 40 mg L−1 as CaCO3, while the permeate water was

In this study we seek to determine the relationship between fractal dimension, the presence of vegetation, and pedestrian perception of streetscape in Taipei, Taiwan. We provide a fractal analysis of street vistas present in Taipei, calculate the amount of vegetation visible in the streets, and assess the perception of the visual quality characteristic of those streets. Correlations between the resultant fractal dimensions, the physical characteristics of the streets, and scores for a single measure of perceived visual quality are discussed and compared with the results from two similar British case studies. The key findings are that judgments of the visual quality of streetscapes are influenced by the presence of vegetation, but they are influenced more by changes in fractal dimension. Vegetation is shown have a strong, positive, and significant correlation with perceptions of variety, coherence, beauty, interest, and preference. Higher levels of vegetation were found in views that were judged as varied, coherent, beautiful, interesting, and likeable. The presence of visible sky and buildings in a view have negative correlations with judgments of variety and interest. The presence of visible boundaries, vehicles, signage, and street furniture all have strong positive correlations with judgments of complexity; conversely, they have strong negative correlations with judgments of order, coherence, and beauty. In the UK cases complexity was correlated positively with perceptions of visual quality, but in the Taiwanese case it was correlated negatively. Keywords: Taiwan, street vista, fractal dimension, visual quality, visual perception, vegetation, urban design

Because there are few undeveloped or underused high-quality water sources in the United States, many utilities are considering impaired water sources to meet increasing demands. Utilities are increasingly expressing interest in developing aquifer recharge projects and desalination technologies. The evolution of those processes offers significant promise to meet future water supply needs. This project investigated the potential to turn wastewater into a high-quality water supply that meets all applicable state and local standards for groundwater recharge to supplement water supplies. The project showed that membranes can meet virtually every regulatory limit imposed, treating the permeate to resolve saturation issues was straightforward, and emerging constituents were removed efficiently.

Fractal analysis offers potential as a tool for evaluating the visual qualities of street vistas. In this paper we outline the concept of fractal dimension as a measure of texture in images of built form and describe a methodology for comparing fractal dimension with perceptions of visual quality in street vistas. We find that a positive relationship exists between levels of fractal dimension and judgments of visual quality in street vistas and outline the applications that this relationship may have.

A new strategy to produce stable barcodes using biological self-assembly of streptavidin- and biotin-functionalized quantum dots is reported. Such systems are of potential use in multiplexed immunoassay and nucleic acid hybridization assays.

The eighteen month occupation of Haddington 1548–1549 is said to be Scotland's longest siege. French, German and Scottish armies battered the defences of the burgh town trying to dislodge a stubborn and forlorn English garrison. In September 1549, the English relief column arrived unopposed and extricated the remaining plague-ridden defenders, leaving the fortifications to be levelled by the besiegers. The fortifications at Haddington were the first appearance in Scotland of the Trace Italienne, designed to defeat conventional siege tactics; bombardment, sapping and storming. Despite long-held assumptions that the fortifications are now lost to history, recent research indicates the survival of elements of the defences.

We report the ability to move and arrange patterns of live embryonic stem cells using holographic optical tweezers. Single cell suspensions of mouse embryonic stem cells were manipulated with holographic optical tweezers into a variety of patterns including lines, curves and circles. Individual cells were also lifted out of the sample plane highlighting the potential for 3D positional control. Trypan blue dye exclusion and Live/Dead™ staining (CMFDA−1, EthHD−1) showed that the cells were still viable after manipulation with the optical tweezers. The ability to move individual stem cells into specific, pre-defined patterns provides a method to study how arrangement and associated small-scale interactions occur between neighbouring cells.

Time-resolved specular neutron reflectivity measurements are presented and interpreted for electroactive polyvinylferrocene (PVF) films subject to potentiodynamic electrochemical control. New data acquisition methodology allows an effective measurement time scale on the order of seconds, which is an improvement over conventional methodology by 2 to 3 orders of magnitude. Reflectivity profiles were obtained for PVF films exposed to aqueous 0.1 M NaClO4 in which PVF films are thermodynamically permselective, with contrast variation via H2O and D2O. Irrespective of any model, the raw profiles show chemically reversible film "breathing" due to redox-driven solvent entry and exit during polymer oxidation and reduction, respectively. Modeling reveals three compositionally distinct regions within the polymer film: interfacial regions at the electrode and solution interfaces and a "bulk" interior. The new methodology, supported by simultaneous in situ visible transmission spectroscopy, reveals an unprecedented level of insight into the temporal and spatial mechanistic details of film solvation changes, including a two-stage (de)solvation mechanism for redox switching, differences in interior (in)homogeneity for reduced and oxidized films, and permselectivity failure under dynamic electrochemical conditions for the reduced (but not oxidized) state, in contrast to static conditions that allow permselectivity for both states.

Fractal analysis can provide a synthetic measurement of place complexity and thereby allow a numerical characterization of places. A fractal analysis of street edges is provided, linking the calculation of fractal dimension to the presence of the physical features making up a street edge. A technique for calculating street edge fractal dimensions is presented and speculation on the use of fractal analysis in comparing the character of differing places is made.

Microchannels (40- microm wide, 10- microm high, 10-mm long, 70- microm pitch) were patterned in the silicone elastomer, polydimethylsiloxane on a microscope coverslip base. Integrated within each microchamber were individually addressable stimulation electrodes (40- microm wide, 20- microm long, 100-nm thick) and a common central pseudo-reference electrode (60- microm wide, 500- microm long, 100-nm thick). Isolated rabbit ventricular myocytes were introduced into the chamber by micropipetting and subsequently capped with a layer of mineral oil, thus creating limited volumes of saline around individual myocytes that could be varied from 5 nL to 100 pL. Excitation contraction coupling was studied by monitoring myocyte shortening and intracellular Ca(2+) transients (using Fluo-3 fluorescence). The amplitude of stimulated myocyte shortening and Ca(2+) transients remained constant for 90 min in the larger volume (5 nL) configuration, although the shortening (but not the Ca(2+) transient) amplitude gradually decreased to 20% of control within 60 min in the low volume (100 pL) arrangement. These studies indicate a lower limit for the extracellular volume required to stimulate isolated adult cardiac myocytes. Whereas this arrangement could be used to create a screening assay for drugs, individual microchannels (100 pL) can also be used to study the effects of limited extracellular volume on the contractility of single cardiac myocytes.

Fractal analysis and the calculation of fractal dimension offers the potential for the numerical characterization of places by providing a synthetic measurement of place complexity. This paper provides a fractal analysis of street-scale urban skylines, linking the calculation of fractal dimension to the presence of the physical features making up a skyline. A technique for calculating skyline fractal dimensions is presented and suggestions are made about the use of fractal analysis in comparing the character of places.

In situ neutron reflectivity measurements have been made on spin cast polyvinylferrocene (PVF) films maintained under electrochemical potential control. The data show that the interiors of both oxidized and reduced films are close to homogeneous. The results show that neutron reflectivity measurements, including isotopic substitution, can provide novel insights into film composition and structure at the electrode/polymer/solution interface. Isotopic substitution of the solvent has allowed the contributions of the solvent and the solute to the total film composition to be separated. Responses were determined to variations in the imposed potential (a constraint at the electrode/polymer interface) and the electrolyte concentration (a constraint at the polymer/solution interface). Solvent entry occurs upon oxidation, resulting is a more diffuse polymer/solution interface. Only at very high concentrations is salt permeation into PVF films sufficiently high to be considered as a possible source of co-ions for r...

We describe a method, based on X-ray photoelectron spectroscopy (XPS) measurements, to assess the extent of protein adsorption or binding on a variety of different muTAS and biosensor interfaces. Underpinning this method is the labeling of protein molecules with either iodine- or bromine-containing motifs by using protocols previously developed for radiotracer studies. Using this method, we have examined the adsorption and binding properties of a variety of modified electrodeposited polymer interfaces as well as other materials used in muTAS device fabrication. Using polymer interfaces modified with poly(propylene glycol) (PPG) chains, our results indicate that a chain of at least approximately 30 monomer units is required to inhibit nonspecific adsorption from concentrated protein solutions. The XPS methodology was also used to probe specific binding of avidins and enzyme conjugates thereof to biotinylated and mixed biotin/PPG-modified polymer interfaces. In one example, using competitive binding, it was established that the mode of binding of a peroxidase-streptavidin conjugate to a biotinylated modified polymer interface was primarily via the streptavidin moiety (as opposed to nonspecific binding via the enzyme conjugate). XPS evaluation of nonspecific and specific peroxidase-streptavidin immobilization on various functionalized polymers has guided the design and fabrication of functionalized interdigitated electrodes in a biosensing muTAS device. Subsequent characterization of this device using scanning electrochemical microscopy (SECM) corroborated the adsorption and binding previously inferred from XPS measurements on macroscale electrodes.

We exploit the mechanical action of surface acoustic waves (SAW) to differentially lyse human cancer cells in a chemical-free manner. The extent to which cells were disrupted is reported for a range of SAW parameters, and we show that the presence of 10 μm polystyrene beads is required to fully rupture cells and their nuclei. We show that SAW is capable of subcellular fractionation through the chemical-free isolation of nuclei from whole cells. The concentration of protein was assessed in lysates with a sensitive microfluidic antibody capture (MAC) chip. An antibody-based sandwich assay in a microfluidic microarray format was used to detect unlabeled human tumor suppressor protein p53 in crude lysates, without any purification step, with single-molecule resolution. The results are digital, enabling sensitive quantification of proteins with a dynamic range >4 orders of magnitude. For the conditions used, the efficiency of SAW-induced mechanical lysis was determined to be 12.9% ± 0.7% of that for conventional detergent-based lysis in yielding detectable protein. A range of possible loss mechanisms that could lead to the drop in protein yield are discussed. Our results show that the methods described here are amenable to an integrated point-of-care device for the assessment of tumor protein expression in fine needle aspirate biopsies.

Bacteria persistence is a well-known phenomenon, where a small fraction of cells in an isogenic population are able to survive high doses of antibiotic treatment. Since the persistence is often associated with single cell behaviour, the ability to study the dynamic response of individual cells to antibiotics is critical. In this work, we developed a gradient microfluidic system that enables long-term tracking of single cell morphology under a wide range of inhibitor concentrations. From time-lapse images, we calculated bacterial growth rates based on the variations in cell mass and in cell number. Using E. coli and Comamonas denitrificans to amoxicillin inhibition as model systems, we found the IC50 determined via both methods are in a good agreement. Importantly, the growth rates together with morphological dynamics of individual cells has led to the discovery of a new form of persistence to amoxicillin. Normal cells that are sensitive to amoxicillin gain persistence or recover from the killing process, if they have had an opportunity to utilise the cytoplasm released from lysed cells close-by. We term this acquired persistence in normal growing cells “opportunistic persistence”. This finding might shed new insights into biofilm resistance and the effect of antibiotics on environmental microbes.

In this article we describe and analyze the emerging audio piracy (MP3) subculture on the Internet. We present the first sociological analysis of this system - a `virtual community' that is revolutionizing the production of popular music. We seek to explain not only the internal social structure of the MP3 community, but also strive to understand how the entire global network of musical distribution and production may be affected by its activity. Particular attention is paid to the role structure that operates within the audio piracy subculture, issues of status and power, the division of labor, as well as questions of social conflict and resolution. The conclusion offers some speculations concerning the impact of MP3 technology on the trajectory of popular music.

Aspartic proteinases (AP) have been widely studied within the living world, but so far no plant AP have been structurally characterized. The refined cardosin A crystallographic structure includes two molecules, built up by two glycosylated peptide chains (31 and 15 kDa each). The fold of cardosin A is typical within the AP family. The glycosyl content is described by 19 sugar rings attached to Asn-67 and Asn-257. They are localized on the molecular surface away from the conserved active site and show a new glycan of the plant complex type. A hydrogen bond between Gln-126 and Manβ4 renders the monosaccharide oxygen O-2 sterically inaccessible to accept a xylosyl residue, therefore explaining the new type of the identified plant glycan. The Arg-Gly-Asp sequence, which has been shown to be involved in recognition of a putative cardosin A receptor, was found in a loop between two β-strands on the molecular surface opposite the active site cleft. Based on the crystal structure, a possible mechanism whereby cardosin A might be orientated at the cell surface of the style to interact with its putative receptor from pollen is proposed. The biological implications of these findings are also discussed.

Creating patterns of biomolecules and cells has been applied widely in many fields associated with the life sciences, including diagnostics. In these applications it has become increasingly apparent that the spatiotemporal arrangement of biological molecules in vitro is important for the investigation of the cellular functions found in vivo. However, the cell patterning techniques often used are limited to creating 2D functional surfaces on glass and silicon. In addition, in general, these procedures are not easy to implement in conventional biological laboratories. Here, we show the formation of a living poly(ethylene glycol) (PEG) layer that can be patterned with visible light on plastic surfaces. This new and simple method can be expanded to pattern multiple types of biomolecule on either a previously formed PEG layer or a plastic substrate. Using common plastic wares (i.e., polyethylene films and polystyrene cell culture Petri-dishes), we demonstrate that these PEG-modified surfaces have a high resistance to protein adsorption and cell adhesion, while at the same time, being capable of undergoing further molecular grafting with bioactive motifs. With a photomask and a fluid delivery system, we illustrate a flexible way to immobilize biological functions with a high degree of 2D and 3D spatial control. We anticipate that our method can be easily implemented in a typical life science laboratory (without the need for specialized lithography equipment) offering the prospect of imparting desirable properties to plastic products, for example, the creation of functional microenvironments in biological studies or reducing biological adhesion to surfaces.

Stroke medicine is a multifaceted specialty that offers opportunities to work in acute settings and help patients in their long term rehabilitation

We have demonstrated a monolithic integrated arrayed waveguide grating (AWG) microspectrometer microfluidic platform capable of fluorescence spectroscopic analysis. The microspectrometer in this proof of concept study has a small (1 cm × 1 cm) footprint and 8 output channels centred on different wavelengths. We show that the signals from the output channels detected on a camera chip can be used to recreate the complete fluorescence spectrum of an analyte. By making fluorescence measurements of (i) mixed quantum dot solutions, (ii) an organic fluorophore (Cy5) and (iii) the propidium iodide (PI)-DNA assay, we illustrate the unique advantages of the AWG platform for simultaneous, quantitative multiplex detection and its capability to detect small spectroscopic shifts. Although the current system is designed for fluorescence spectroscopic analysis, in principle, it can be implemented for other types of analysis, such as Raman spectroscopy. Fabricated using established semiconductor industry methods, this miniaturised platform holds great potential to create a handheld, low cost biosensor with versatile detection capability.

The 2014 CCP4 Study Weekend, held at the University of Nottingham, covered the subject of complementary methods. While CCP4 meetings generally cycle through the main crystallography software topics of data processing, phasing, molecular replacement and refinement, memorable meetings in the past were also held on topics such as macromolecular complexes (2006) and low-resolution structure determination (2008). The topic of the 2014 meeting reflected the fact that various methods can complement the traditional crystallographic approaches in order to shed light on the dynamics, inter­actions and higher order structures of the macromolecules on which we work. In addition, these complementary techniques can also assist us in the process of protein structure determination.The biological systems we study are invariably multi-component and are involved in complex dynamic processes; indeed more than half of the macromolecules deposited in the PDB are oligomeric. In recent years, the extraordinary developments that have taken place in the techniques available in molecular biophysics provide macromolecular crystallographers with a strong driving force to integrate their results into a more complete understanding of the systems under study. Whilst NMR (nuclear magnetic resonance) and EM (electron microscopy) are two of the classic complementary methods, today spectroscopic, scattering, calorimetric and numerous other techniques are commonly used in our laboratories. Likewise, synchrotron beamlines today have advanced from being simple producers of bright X-rays to experimental set-ups with multiple on-line detectors, and synchrotron facilities have become major cross-disciplinary science hubs with a multitude of methods and equipment available. CCP4 is therefore rightly engaging with developers of these important techniques, which augment our work, to fully exploit their potential as tools for the structural biologist.The first session of the meeting was on biophysics, and was chaired and introduced by Mike Hough (Essex). A fascinating description by Huaying Zhao (NIH, Bethesda) covered new computational methods for combining various biophysical measurements in the analysis of multi-component protein complexes. This was followed by a presentation on the application of several such methods by Ehmke Pohl (Durham) and a review and presentation of software for in crystallo spectroscopic techniques by Florian Dworkowski (SLS). Antoine Royant (ESRF) then gave an insightful presentation on the capabilities of the ESRF Cryobench facility for various spectroscopic investigations of protein crystals.The next session, which was chaired by Edward Snell (Hauptman–Woodward Institute), was concerned with optimal strategies for the collection of solution scattering data – measurements that allow description of protein and other macromolecular assemblies. A presentation from Javier Pérez (Soleil) described the application of these methods to membrane proteins embedded in lipid/detergent. In the subsequent two presentations, Frank Gabel (IBS Grenoble) discussed the uniqueness of model predictions obtained by SAXS techniques and Adam Round (ESRF) described a software tool for automated SAXS data collection and online feedback.It is evident that the availability and recent developments in these methods challenge the crystal-centred way in which we work, as it is essential to understand what’s in that crystal. It was therefore logical to address the question of how we presently bring our samples into the X-ray beam. In the session on crystal manipulation, chaired by Frank von Delft (Diamond/Oxford), Alex Soares (BNL) outlined fascinating methods for mounting crystals and simultaneous ligand-screening, involving use of acoustic droplets. Alternative approaches for fully automated crystal mounting were then described by Florent Cipriani (EMBL, Grenoble). Next, Joseph Lyons (Aarhus) described methods for automatically locating microcrystals at the synchrotron beamline and Dianfan Li (Dublin) told us about an application of serial femtosecond crystallography using the free-electron laser to study membrane proteins crystallized in the lipid cubic phase (LCP). Finally, Robin Owen (Diamond) gave a presentation on room temperature in situ data collection. While the session focused on presently available tech­niques at synchrotron radiation sources, it is clear that free-electron laser applications will change the way in which we look at crystal mounting, as they provide the opportunity to add time-resolution to the crystallographic experiment.The classic NMR and EM methods were presented on day two of the meeting, and progress on the respective CCPs is covered in this issue. The session on EM was chaired and introduced by Helen Saibil (Birkbeck, London) who also reported on a new facility at the Diamond Light Source, providing 24/7 access to EM modelled on the crystallography BAG system (Block Allocation Group). A fascinating account of the theory and practice of EM studies of heterogeneous macromolecular complexes was presented by Elena Orlova (also Birkbeck). Following this, Werner Kühlbrandt (MPI, Frankfurt) gave an intriguing and wide-ranging lecture on the applications of single-particle imaging, molecular tomography and high-resolution cryo-EM. In his words (‘watch out!’), these herald a new phase of EM where we can expect to see a revolution in resolution because of new detector technology, similar to what has happened over the last ten years in the X-ray field with the advent of solid-state detectors and millisecond exposures. Alan Brown (MRC, Cambridge) reported on methods in ribosome structure determination for the interpretation of high-resolution EM data, as the borders between EM and X-ray structures now dissolve and X-ray crystallographic software can be used in EM refinement.The subsequent session on protein dynamics was chaired by Arwen Pearson (Leeds/Hamburg) and included a presentation by John Christodoulou (UCL) on the unique insights provided by NMR spectroscopy on the dynamics, disorder and meta-stability of proteins. Next up, Fraser MacMillan (UEA) presented a number of applications of electron paramagnetic resonance in studying metalloproteins and spin-labelled macromolecular assemblies. Finally, the methods available to study the molecular dynamics of biomacromolecules were presented by Sarah Harris (Leeds), who demonstrated that these approaches, in the same way as experimental biophysical methods, powerfully complement structure determination.The final session dealt with a different aspect of providing complementary structural information. Since nearly three quarters of entries in the PDB contain a ligand and indeed the atomic description for many structures could well be incomplete as these most likely contain unknown ligands, we initially thought we might address this, but instead decided to focus on the subject of drug design, which was introduced and chaired by Dave Brown (Kent). Following on from the previous topics on biophysical methods and NMR, the presentation by Glyn Williams (Astex) included the applications of NMR in drug screening. This was followed by a description of approaches for the identification of binding pockets in proteins and ligand screening by Judit Debreczeni (Astra Zeneca). The session concluded with a demonstration of some of the related features of the new CCP4 GUI by Martin Noble (Newcastle).As organizers, we were aware of the time constraints in a two-day meeting and we can perhaps partially redress the necessary limited selection of topics by touching briefly on a number of other complementary techniques in the structural biologist’s armory. One feature of X-ray diffraction is that hydrogen atoms are nearly always poorly defined in the final structure due to the low scattering factor of this element. In contrast, neutrons have a much higher cross section and thus are far more sensitive to hydrogen than X-rays, and the isotope of hydrogen, deuterium, scatters neutrons as strongly as does carbon. Deuterium can be introduced into protein crystals by vapour diffusion or soaking and non-exchangeable hydrogen atoms can be replaced by expressing the protein in deuterated growth media, using techniques which are largely routine in the NMR field. Along with improvements in detectors and pulsed sources, these techniques have allowed neutron diffraction data to be collected from much smaller crystals than ever before, thus shedding important mechanistic light on a number of enzymes. Another technique, which has allowed studies of catalytic mechanisms is the X-ray Laue method, due to the speed with which the data can be collected following initiation of the reaction. Recent advances in this field include the study of laser-triggered reactions where the light pulses can be synchronized with the synchrotron beam by use of single-bunch mode and/or high-speed X-ray beam choppers. Finally, an open question not addressed in the meeting is how we deposit all these complementary data with our structure.We would like to wholeheartedly compliment the speakers and chairs for their excellent contributions to all sessions of the 2014 Study Weekend. We especially thank those who contributed papers to the current journal issue, which, we hope, constitutes an informative record of a very memorable meeting. We also thank the CCP4 administrative staff (Karen McIntyre, Carol Malpass and Shirley Miller) for their pivotal support in the organization of the meeting.

We demonstrate a single-step method for the generation of collagen and poly-l-Lysine (PLL) micropatterns on a poly(ethylene glycol) (PEG) functionalized glass surface for cell based assays. The method involves establishing a reliable silanization method to create an effective non-adhesive PEG layer on glass that inhibits cell attachment, followed by the spotting of collagen or PLL solutions using non-contact piezoelectric printing. We show for the first time that the spotted protein micropatterns remain stable on the PEG surface even after extensive washing, thus significantly simplifying protein pattern formation. We found that adherence and spreading of NIH-3T3 fibroblasts was confined to PLL and collagen areas of the micropatterns. In contrast, primary rat hepatocytes adhered and spread only on collagen micropatterns, where they formed uniform, well defined functionally active cell arrays. The differing affinity of hepatocytes and NIH-3T3 fibroblasts for collagen and PLL patterns was used to develop a simple technique for creating a co-culture of the two cell types. This has the potential to form structured arrays that mimic the in vivo hepatic environment and is easily integrated within a miniaturized analytical platform for developing high throughput toxicity analysis in vitro.